When light is directed onto a molecule, the vast majority of the incident photons are elastically scattered without a change in frequency. This is termed Rayleigh scattering. However, the energy of some of the incident photons (approximately 1 in every 107 incident photons) is coupled into distinct vibrational modes of the molecule's bonds. Such coupling causes some of the incident light to be inelastically scattered by the molecule with a range of frequencies that differ from the range of the incident light. This is termed the Raman effect. By plotting the frequency of such inelastically scattered light against its intensity, the unique Raman spectrum of the molecule or material under observation is obtained. Analysis of the Raman spectrum of an unknown sample can yield information about the sample's molecular composition.
The intensity of Raman scattered radiation can be significantly increased by bringing the Raman-active molecule(s) or materials in very close proximity (for example ≦20 Å) to certain types of nanostructured metal surfaces. For example, molecules adsorbed to roughened gold, silver or copper or other free electron metal surfaces can experience million-fold increases in Raman intensity. This effect, called surface enhanced Raman scattering and alternately surface enhanced Raman spectroscopy (in both cases referred to as SERS) can be observed with metal colloidal particles, metal films on dielectric substrates, with ordered or disordered metal particle arrays, and on a variety of other nanoscopic, mesoscopic, microscopic, or macroscopic surfaces.
The mechanism by which SERS or similar surface enhanced spectroscopic (SES) phenomena occur is understood, and is thought to result from a combination of electromagnetic (e.g. local field) and chemical (e.g. charge transfer) effects. Representative SES techniques include, but are not limited to surface enhanced Raman spectroscopy (SERS) and surface enhanced resonance Raman spectroscopy (SERRS). In SERS or SERRS, the metal or other enhancing surface will couple electromagnetically to incident electromagnetic radiation and create a locally amplified electromagnetic field that leads to 102- to 109-fold or greater increases in the Raman scattering of a SERS active molecule situated on or near the enhancing surface.
As noted above, Raman scattering occurs when a molecule is illuminated with incident light. Typically, the incident light source is a monochromatic laser. The correct selection of the incident laser wavelength can be an important consideration for Raman spectroscopy. For instance, many samples, especially those of an organic or biological nature will be (or contain) fluorescent species. Exciting these samples with a laser in the green (532 nm) may promote fluorescence, which will swamp any underlying Raman spectrum to such an extent that it is no longer detectable.
Accordingly, Raman spectroscopy techniques such as SERS and SERRS often feature the use of a laser in the red, for example, 633 nm or near infrared (NIR), for example, 785 nm or 1064 nm portions of the spectrum. Since these sources have somewhat lower photon energy that green or shorter wavelength sources, a red or NIR laser may not promote the electronic transitions which will resulting in fluorescence, resulting in Raman scattering which is easier to detect.
Unfortunately, as the incident wavelength is increased, from green to red to NIR, the efficiency of Raman scattering decreases, since it varies as (1/wavelength)4. Therefore, longer integration times and/or higher power lasers are necessary to acquire a suitable signal as the incident wavelength increases. Typically, lasers emitting light having a wavelength of no longer than about 1064 nm are used for Raman spectroscopy. Longer wavelengths result in such diminished scattering efficiency that the use of longer wavelength sources is impractical.
Certain safety concerns are present when using shorter wavelength lasers. In particular, lasers emitting at 1064 nm, 785 nm and shorter wavelengths are potentially hazardous if directed into a human eye. The maximum eye-safe exposure to laser light increases dramatically at 1400 nm and longer wavelengths as shown in the graphs 100 and 102 of FIG. 1, taken from the American National Standards Institute (ANSI) Standard Z136.1-2007. Accordingly, Raman microscopes or Raman spectrometers which use lasers emitting at 1064 nm or shorter wavelengths must be either stationary devices that cannot inadvertently illuminate a technician's eye, or these devices must include complex safety mechanisms to avoid the possibility of accidental damage to a technician's eye. Safety concerns thus limit the feasibility of hand held or other laser sources which may be implemented in a variety of uncontrolled, non-laboratory situations. Longer wavelength radiation can be relatively eye-safe; however as described above, longer wavelength light sources result in inefficient scattering using known enhancement strategies.
Generally, the suitability of a particle-based enhancing surface for use at longer wavelengths can be improved by making the particle larger, because larger particles typically exhibit plasmon bands shifted further into the NIR relative to smaller particles. Many SES active taggants are designed for use or dispersion in fluids, and thus are best implemented with particles or tags that are sized and have a suitable density to remain suspended in the selected liquid without settling. Therefore, the design of particles that may be interrogated at longer, eye-safe wavelengths and are also suspendable in a liquid is particularly problematic.
The present invention is directed toward overcoming one or more of the problems discussed above.